Project summary (1-2 para): Nicotinic acetylcholine receptors are ligand-gated ion channels that are widely expressed throughout the central and peripheral nervous systems. The ?4?2 nicotinic receptor is the most abundant subtype in the brain and is linked to nicotine addiction and neurological disorders including Alzheimer's and Parkinson's diseases. Expression of the ?4?2 receptor is upregulated by chronic exposure to nicotine in vitro and in the brains of smokers compared to that of non-smokers. Accordingly, there have been extensive efforts to develop small molecules to reduce tobacco addiction by targeting this receptor type, but these efforts are hampered by the limited amount of structural information for nicotinic receptors. Atomic structures of a desensitized-state nicotinic receptor in detergent obtained by X-ray crystallography and Cryo-EM are known, but high-resolution structures in a physiologically relevant lipidic environment remain unresolved. Moreover, we lack structural information for this receptor in the different states along the gating cycle. Here I propose to first identify combinations of lipids that support robust channel function using electrophysiological approaches. In parallel I will optimize sample preparation for the ?4?2 nicotinic receptor in lipidic nanodiscs, leveraging the information from the functional studies in defined lipidic environments. I will use these preparations to obtain high resolution structural information for the ?4?2 nicotinic receptor in distinct conformational states, in an experimental condition tied directly to a physiological preparation. Structural information for the receptor in a membrane and in different conformational states will provide touchstones for understanding allosteric gating of these receptors and templates for development of state-selective pharmacological tools and therapeutics.